Locus coeruleus lesions and PCOS: role of the central and peripheral sympathetic nervous system in the ovarian function of rat.

BACKGROUND
"Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction". "Autonomic and central nervous systems play important roles in the regulation of ovarian physiology". The noradrenergic nucleus locus coeruleus (LC) plays a central role in the regulation of the sympathetic nervous system and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway and its activation is essential to trigger spontaneous or induced LH surges. This study evaluates sympathetic outflow in central and peripheral pathways in PCO rats.


OBJECTIVE
Our objectives in this study were (1) to estimate LC activity in rats with estradiol valerate (EV)-induced PCO; (2) to antagonized alpha2a adrenoceptor in systemic conditions with yohimbine.


MATERIALS AND METHODS
Forty two rats were divided into two groups: 1) LC and yohimbine and 2) control. Every group subdivided in two groups: eighteen rats were treated with estradiol valerate for induction of follicular cysts and the remainders were sesame oil groups.


RESULTS
Estradiol concentration was significantly augmented by the LC lesion in PCO rats (p<0.001), while LC lesion could not alter serum concentrations of LH and FSH, like yohimbine. The morphological observations of ovaries of LC lesion rats showed follicles with hyperthecosis, but yohimbine reduced the number of cysts, increased corpus lutea and developed follicles.


CONCLUSION
Rats with EV-induced PCO increased sympathetic activity. LC lesion and yohimbine decreased the number of cysts and yohimbine increased corpus lutea and developed follicles in PCO rats.


Introduction
t is estimated that 6-7% of women of reproductive age have polycystic ovarian syndrome (PCOS) (1). Polycystic ovary syndrome (PCOS), the most common female endocrine disorder, is a complex and heterogenic disease with unknown etiology. PCOS is characterized by reproductive disturbances including chronic anovulation, hyper-androgenism, and polycystic ovaries (2). Autonomic and central nervous systems play important roles in the regulation of ovarian physiology (3).
"It is well known that one of the major neurotransmitters that control LH secretion is norepinephrine (NE)". The NE turnover in the preoptic area parallels changes in circulating LH concentrations (4). Infusion of noradrenaline intracerebroventricularly (5) in the rat, suppresses LH pulses, as does electrical stimulation of the brain stem ascending noradrenergic pathways (6), "thus suggesting that increases in noradrenergic activity results in a suppression of the GnRH pulse generator". In contrast, peripheral administration of -adrenergic receptor antagonists decreases the frequency of the GnRH pulse generator (7). The finding that both reduction and increase in adrenergic receptor activity have the same effect on pulsatile LH secretion led Leng and colleagues (8) "to propose that fluctuating patterns of adrenergic receptor activity are essential for pulsatile GnRH release, a postulate substantiated by theoretical modeling experiments". The syndrome is associated with peripheral and I central factors that influence sympathetic nerve activity. Thus, the sympathetic nervous system may be an important factor in the development and maintenance of PCOS (9).
"Although the involvement of the sympathetic nervous system has been suggested in PCOS, this is the first time that direct intraneural recordings of sympathetic nerve activity have been obtained in women with PCOS". PCOS is associated with hyperandrogenemia, hyperinsulinemia (10), and insulin resistance, as well as abdominal obesity, cardiovascular disease and obstructive sleep apnea (11), all factors hypothesized to be associated with increased activity of the sympathetic nervous system (10).
Increasing evidence also supports the presence of a primary defect in ovarian and adrenal steroidogenesis in PCOS (12), resulting in elevated androgen production, both basally and in response to LH (13). Women with PCOS have significantly higher sympathetic nerve activity than their matched controls and the increased sympathetic outflow is related to hormonal and metabolic features (14).
"The locus coeruleus (LC), a welldelineated cluster of noradrenaline-containing neurones located adjacent to the fourth ventricle in the pontine brainstem is the major noradrenergic nucleus in the brain. The LC activity is remarkably synchronized, producing a coordinated release of noradrenaline (NA) throughout the central nervous system." It is estimated that ~50% of all of the noradrenergic projections in the central nervous system originate in the LC (15) and several brainstem catecholaminergic neurons are known to innervate the sympathetic preganglionic neurons and thereby are potential modulators of the sympathetic nervous system response to stress (16,17). For example, chronic stress promotes PCO in rats, which seems to be initiated by an increased central noradrenergic tonus dictated by an augmentation in the LC activity (18).
Electrolytic lesions of the LC block the preovulatory surge of LH. This blockade of LH surge is accompanied by a decrease in the NE content in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) (19). These findings strongly suggest that NE from the LC plays an important role mediating the positive feedback action of E 2 on LH secretion (20).
Experimental induction of a polycystic ovarian syndrome (PCOS) in rodents by the administration of a single dose of estradiol valerate (EV) results in activation of the peripheral sympathetic neurons that innervate the ovary. This activation is evidenced by an increased capacity of ovarian nerve terminals to incorporate and release norepinephrine (NE), an increase in ovarian NE content, and a decrease in ovarian beta-adrenergic receptor number in the ovarian compartments receiving catecholaminergic innervation.
This increased ovarian sympathetic outflow suggested by these alterations in catecholamine homeostasis was accompanied by a thecal cell-interstitial tissue selective down-regulation of beta-adrenergic receptors; the beta-adrenergic receptor concentration in these sympathetically innervated ovarian compartments was significantly lower in PCO than during the estrous phase of the estrous cycle, a time at which the beta-adrenergic receptor concentration reaches its lowest levels in normal cycling ovaries (21).
The sympathic ovary nerve (SON) transection also reduced the elevated levels of ovarian NE resulting from EV treatment and caused up-regulation of beta-adrenoreceptors. Most importantly, SON transection restored estrous cyclicity and ovulatory capacity. The results indicate that the increased output of ovarian steroids in PCOS is at least in part due to an enhanced responsiveness of the gland to both catecholaminergic and gonadotropin stimulation.
The ability of SON transection to restore a normal response indicates that the alteration in steroid output results from a deranged activation of selective components of the noradrenergic innervation to the ovary. These findings support the concept that an alteration in the neurogenic control of the ovary contributes to the etiology of PCOS (22).
The aim of this study was to evaluate the effects of LC (central) lesion and yohimbine (peripheral) in modeling of PCOS in rat, which occurs during estrus phase.

Animals and care
"Adult female Wistar rats weighing 220-230 g (7-8 wk of age) from the animal house of the Pastor Institute were kept in a central animal care facility under a 12-h light, 12-h dark cycle and controlled temperature (24±0.5 o C). Food and water were provided ad libitum".
Vaginal smears were taken daily and only rats showing estrous phase were used in the experiment. Study groups (forty two rats) were divided into two groups: 1) LC and yohimbine, 2) control (sesame oil) and intact group. Every group subdivided in two groups: eighteen rats were treated with estradiol valerate for induction of follicular cysts (PCO modeling) and the remainders were sesame oil groups.
All of the animal studies were also approved by the Ethics Committee of Tehran University of Medical Sciences and experiments were performed in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (publication No. 85-23, revised 2007).

Experimental design Vaginal smear
Estrous cyclicity was monitored by vaginal smears obtained between 0800 and 1200 hours, and it was assessed by analysis at the light microscopy level of the relative proportion of leukocytes, epithelial and cornified cells found in daily vaginal lavages, which characteristically change during different stages of the estrous cycle. The rat estrous cycle (estrus, diestrus1, diestrus2, and proestrus) usually lasts about 4 days, in controls or PCO rats (23).

Hormonal treatment and study procedure
After 1 week of acclimatization, smear is taken from 7-8 week-old rats (n=24) and after 4 days each rat receives an i.m. injection of Estradiol Valerate ( Aburaihan Co., Iran), 2mg in 0.2 ml of seasame oil, to induce PCO as described by Brawer 1996 (24).
Control rats (n=6) were injected with sesame oil. "All experiments were performed 60 days after the injection, when follicular cysts are first detected". Then PCO rats and sesame oil groups were subdivided in two groups: LC lesion and without LC lesion (normal LC) groups. The dose of yohimbine (Yoh) in this study was 0.4mg/kg according to dose response trial in two groups: Se-Yoh and Est-Yoh. Vaginal smears were taken daily to verify estrous cycle regularity, food and water were provided ad libitum.

LC neurochemical lesion
Under ketamine 100 mg/kg body weight, ip and xylazine 14 mg/kg body weight, ip anesthesia, rats were positioned in a stereotaxic instrument with the incisor bar set at the zero point. The dorsal surface of the skull was exposed, and holes, 2 mm in diameter, were drilled bilaterally. Bilateral LC lesions were made using an SEG 5 gl syringe.

Measurement of circulating levels of gonadotropins and gonadal steroid
Blood samples were collected from the heart and centrifuged at 3500g for 10 min. Serum was isolated and immediately frozen at -80°C. Serum luteinising hormone (LH),

Ovarian morphology
The ovaries from controls (EV-treated), LC lesion and yohimbine treated were removed, cleaned of adherent connective fat tissue, and fixed in 10% formaldehyde buffer for at least 24 hours. Ovaries were imbedded in paraffin, cut in 8-µm sections, and stained with hematoxylin and eosin.

Statistical analysis
Data are presented as mean±SEM. Statistical differences were determined by two-way and one-way ANOVA followed by the Bonferroni post hoc test. SPSS-13 was used for data analysis. p<0.05 was considered as significant level.

LC lesion and yohimbine administration effects on plasma levels of ovarian steroids and gonadotropins
The serum level of Estradiol was significantly increased in PCO rat with (p<0.001) and without (p<0.001) LC lesion in comparison with intact group. Also there was a significant difference between serum level of Estradiol in PCO and non PCO rat with LC lesion (p=0.001) (Figure 1).
In yohimbine groups estradiol concentration were significantly increased by systemic administration in comparison with control (p<0.001) and intact group (p<0.001) (Figure 2). No significant changes were noted in the levels of FSH and LH in all groups (Table I and II). In PCO and control (sesame oil) groups of yohimbine there was significantly increasing of estradiol level and corpora lutea.

Histological findings LC lesion and yohimbine administration upon ovarian morphology
The morphological analyses of ovaries from control rats on estrus revealed the presence of numerous corpora lutea in different stages of development and regression, many of which clearly resulted from recent ovulation, as well as some atretic antral follicles ( Figure 5A).
Ovarian morphology on estrus of rats exposed to Estradiol Valerate ( Figure 5B) and LC lesion induced several ovarian morphological alterations, marked predominantly by the presence of numerous healthy antral follicles with small size and follicles with enlarged theca cell layer (hyperthecosis) ( Figure 5C) and cyst.
Ovulation with corpora lutea formation occurred during yohimbine administration together with decreasing of cysts number ( Figure 5D).

Discussion
The aim of this study was to test the hypothesis that sympathetic nervous system has a critical role in creation of poly cystic ovary syndrome modeling in rat. The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate LHs secretion in female rats.
Oestrogen receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. Oestradiol has been reported to regulate the gene expression of noradrenaline biosynthetic enzymes in the LC (26), "but the effects of ovarian steroids on the activity of LC neurones and the relevance of such actions for LH secretion remain poorly understood".
The locus coeruleus (LC) is a densely packed cell group located in the dorsal pons which contains approximately half of all noradrenergic neurons in the rat brain. Females have a higher number of LC neurons than males, and this difference appears to depend on estradiol receptor-β (ER-β) (27).
The neurons arising in the LC, project directly to the GnRH-rich regions of the preoptic area (28) and selective lesioning of the LC results in a decrease in noradrenaline levels within the preoptic area (29), hypothalamus and prevents the occurrence of the LH surge (30). LC noradrenergic neurons may directly alter the activity of the GnRH neural system. Numerous studies implicated noradrenaline as an important regulator of GnRH/LH release.
Infusion of noradrenaline either intracerebroventricularly or directly into the preoptic area of the rat suppresses LH pulses (31). It is interesting to note that near complete interruption of ascending noradrenergic pathways did not alter LH pulse frequency (32), whereas discrete lesions of the LC, destroying 50% or more of the nucleus, resulted in a permanent inhibition of pulsatile LH release (33).
Recently, it was demonstrated that women with PCOS have enhanced ovarian productions of nerve growth factor (NGF), a strong marker of sympathetic nerve activity. These results suggest that overproduction of ovarian NGF is a component of PCO morphology in humans. In a transgenic mouse model over expressing NGF in the ovaries, persistent elevation in plasma LH levels is required for the morphological abnormalities to appear (34). The transection of the superior ovarian nerve in the EV-induced rat PCO model reduces the steroid response, increases β 2 -adrenoceptor expression to more normal levels, and restores estrus cyclicity and ovulation (35).
Also, blockade of endogenous NGF action restores the EV-induced changes in ovarian morphology and expression of the sympathetic markers α 1 -and β 2adrenoceptors, p75 neurotrophin receptor p 75NTR , NGF-tyrosine kinase receptor, and tyrosine hydroxylase. These data confirm the close interaction between NGF and the sympathetic nervous system in the pathogenesis of steroid-induced PCO in rats (36).
In contrast, however, peripheral administration of -adrenergic receptor antagonists also decreases the frequency of the GnRH pulse generator (37). Our data from this study suggest that, LC noradrenergic neurons have a critical role in feedback system of estradiol because its lesion in PCO rats increased estradiol level and induced hyperthecosis (38), that shows LC lesion cause latency in the processing of modeling PCO in rat.
Yohimbine as the α2 receptor antagonist showed increasing estradiol level (like LC lesion) and numbers of corpus lutea and developed follicles for characterizing of the peripheral sympathetic nervous system in PCO rats. It is interesting to note that data of yohimbine shows that in sesame oil group there are increasing of estradiol level and corpus lutea that resembles PCO which seems to be the direct effect of this antagonist in rat ovary.
We did not find changes in plasma LH and FSH levels in either groups of LC and yohimbine. Our results indicate that LC lesion and yohimbine have effectiveness in processing of modeling of PCOS in rat. These findings support the theory that increased sympathetic activity contributes to the development and maintenance of PCOS.